Effects of plant growth regulators and high temperature on colour ...

Journal of Horticultural Science & Biotechnology (2013) 88 (4) 387–392

Effects of plant growth regulators and high temperature on colour development in ‘Crimson Seedless’ grapes By RINAT OVADIA1, MICHAL OREN-SHAMIR1, TATIANA KAPLUNOV2, YOHANAN ZUTAHY2, AMNON LICHTER2 and SUSAN LURIE2* 1 Department of Ornamental Horticulture, The Volcani Center, Agricultural Research Organization, P. O. Box 6, Bet Dagan 50250, Israel 2 Department of Postharvest Science, The Volcani Center, Agricultural Research Organization, P. O. Box 6, Bet Dagan 50250, Israel (e-mail: [email protected]) (Accepted 6 March 2013) SUMMARY ‘Crimson Seedless’ is a red, seedless table grape (Vitis vinifera) variety which may not develop adequate colour during hot weather. This problem can be alleviated, in part, by applying abscisic acid (ABA). We were interested to determine whether a gibberellin (GA3), which is used to improve the size of grape berries, might interfere with this response. We were also interested in examining whether short periods of high temperature would affect the response to ABA. In addition, we assessed the optimum time at which to apply ABA, and the role of ethylene in berry colour development. These experiments were conducted in commercial vineyards in Israel, and on detached berries held under controlled conditions. GA3 had no effect on the response to ABA. There was a mixed response to ethylene, and to the application of ethylene inhibitors in the vineyard or in controlled studies in growth chambers. The best response in terms of colour development (i.e., anthocyanin levels) on detached berries occurred when 200 mg l–1 ABA was applied in early August. A short period of high temperature did not affect the response to ABA, with similar accumulations of anthocyanins at 18ºC, 25ºC, or 38ºC for 24 h after ABA was applied. These results indicate that: (i) the practice of applying a GA3 spray to increase berry size did not affect the berry response to ABA; (ii) the responsiveness of grape berries to ABA was maintained for an extended time after the initiation of veraison; (iii) a period of high temperature after the application of ABA did not compromise the response of detached berries to ABA; and (iv) the response of berries to ethylene was not maintained after harvest. The best treatment to enhance colour in ‘Crimson Seedless’ grapes was therefore 200 – 400 mg l–1 ABA applied 2 – 3 weeks after veraison.

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rimson Seedless’ grapevine (Vitis vinifera) berries often fail to achieve satisfactory red colour in hot climates. In part, this is due to high night temperatures that inhibit the accumulation of anthocyanins (Spayd et al., 2002). The colour of grape berries is determined by the quantity and composition of anthocyanins in the peel. Colour development is influenced by various environmental, physiological, and chemical factors such as temperature, light, and the levels of various plant growth substances (Dokoozlian and Hirschefeld, 1995; Downey et al., 2006). Consequently, canopy and crop management practices that increase light levels can improve berry colour development. Application of the ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA; EthrelTM; Bayer Cropscience, Munich, Germany), can increase the accumulation of anthocyanins and advance ripening (Dokoozlian et al., 1995; Lichter et al., 2004; Szyjewicz and Rosner, 1984). In grape berries, the accumulation of anthocyanins begins at veraison (the onset of maturation). This is related to the interaction between auxins, which decrease anthocyanin concentrations, and abscisic acid (ABA) which increases anthocyanin concentrations (Ban et al., 2003; Lund et al., 2008). Exogenous ABA increased the concentrations of anthocyanins in the berry skins of a *Author for correspondence.

number of inter-specific hybrid grapes (Han et al., 1996; Kataoka et al., 1982; Lee et al., 1997; Matsushima et al., 1989) and in seedless V. vinifera grapes (Cantin et al., 2007; Peppi et al., 2006). Grapes with high concentrations of anthocyanins in their skin are darker and redder than grapes with low concentrations of anthocyanins. However, the relationship between pigment content and colour is not linear. Large increases in anthocyanins may have little effect on colour (Peppi et al., 2006; 2007a). In a previous study, we examined the use of a commercial formulation of ABA (ProToneTM; Valent BioSciences, Libertyville, IL, USA) on red colour development in ‘Crimson Seedless’ grapes in the vineyard and in detached single berries with petioles (Lurie et al., 2009). We found that both systems responded similarly. In vineyards, there was some variation in response with location and season (Peppi et al., 2006; 2007a). These variations could be due to differing horticultural practices, as well as to the weather. Environmental responses are difficult to reproduce in the field, but the effect of temperature can be determined using detached berries in a controlled growth environment. Therefore, in this study, we examined the effect of high temperature applied before or after an ABA dip, on anthocyanin accumulation. We also examined the effects of ABA, gibberellic acid (GA3), and ethylene, alone or in combination, as well as the time of ABA application

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PGRs and anthocyanin accumulation in grape berries

on berry colour development. These experiments were conducted both as treatments on detached berries in the laboratory and by vineyard application.

MATERIALS AND METHODS General vineyard practices Experiments were conducted on ‘Crimson Seedless’ grapevines in three commercial vineyards in the central and southern coastal areas of Israel over three seasons (2009 – 2011). The vines were trained to a Y-shaped bilateral cordon system and trellised, and standard cultural practices were followed. Shoots were thinned to one primary node per shoot when they were 10 cm-long, and the basal leaves and lateral shoots were removed.The standard fruit load was 45 clusters per vine. Treatments in the vineyard were arranged in randomised blocks, with three vines in each block and four blocks per treatment. Handling of detached berries Colour development in detached grape berries was assessed as described by Lurie et al. (2009). Berries with pedicels were sampled from eight-to-ten clusters and were separated into green or breaker-stage berries. Twenty berries per treatment were placed separately in 1.5 ml polypropylene tubes with their pedicels immersed in various solutions. The solutions contained 2% (w/v) sucrose, 0.05% (w/v) PPM (Preservative for Plant Tissue Culture Media; Phyto Technology Laboratory, Washington, D.C., USA) and ABA, as specified. The samples were held for 7 d at 23oC (day) and 15oC (night), followed by measurement of their anthocyanin concentration. Anthocyanin concentrations were measured in grape berry skins (four-to-five berries per sample, with three replications). The pigments were extracted by immersing the skins in 20 ml of 11:5:1 (v/v/v) methanol:water:acetic acid at 4ºC overnight. Anthocyanins were determined by measuring the absorbance of the extract at 530 nm using a UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). Application of gibberellic acid and abscisic acid The effect of GA3 on the response of detached grape berries to ABA was evaluated in two vineyards. In each vineyard, four blocks of three vines were not treated until sampling, while four blocks were sprayed, when the berries were 6 mm in diameter, with GA3 (ProGib; Valent BioSciences) containing 0.025% (v/v) Triton X100 (Sigma, St. Louis, MO, USA) as a surfactant. The spray, directed onto the clusters, was applied at 2,000 l ha–1. Clusters were harvested three times after the start of veraison, and the berries were treated by immersing the pedicels in 0, 200, or 400 mg l–1 ABA (ProTone, Valent BioSciences). Anthocyanin concentrations were determined after 7 d, as specified above. The values for each treatment were pooled over the two vineyards and three sampling times. The effect of GA3 on the response of berries to ABA applied in the vineyard was also tested in two vineyards. GA3 was applied, as specified above, and ABA was applied at 0, 200, or 400 mg l–1 1 week after veraison, when approx. 90% of the clusters began to change colour. The colour of the clusters was assessed 2 weeks later by visual

inspection of 25 consecutive clusters per row for each replicate. Clusters were rated as being green, light (partly green), medium (fully-coloured, but light colour), or dark (intense colour) on most berries in the cluster. The data for colour development are presented as the combined average percentage of medium- and dark-coloured clusters, with four blocks (replicates) per treatment. Application of ethylene and 1-methylcyclopropene The effects of 2-chloroethylphosphonic acid (CEPA; EthrelTM, Bayer Cropscience), ethylene, and the ethylene action inhibitor, 1-methylcyclopropene (1-MCP) on berry colour were examined in two experiments with ten clusters per treatment. In Experiment I, clusters were harvested and treated for 24 h by injecting ethylene (at 100 µl l–1) into a 200 l gas-tight container through a rubber septum, or by dissolving 1-MCP powder (AgroFresh, Midland, MI, USA) in water at 40ºC in a tube placed in the bottom of the container, opening the tube and sealing the container to give 2 µl l–1 1-MCP, or by both treatments. Ten control clusters were held in air for 24 h. Green and breaker-stage berries (n = 20) with pedicels were sampled from each treatment and incubated for 7 d before anthocyanin determinations. This experiment was repeated twice. In Experiment II, 40 clusters were harvested and ten clusters were dipped in CEPA (EthrelTM) at 0, 0.48, 0.96, or 1.44 g l–1 [0.2%, 0.4%, or 0.5% (w/v) respectively]. Breaker-stage berries (n = 20) from the clusters were then detached and incubated with or without 50 mg l–1 ABA. Anthocyanin concentrations were determined after 7 d. To compare the response of berries in the vineyards to CEPA, eight blocks of vines were treated with 0, 200, or 400 mg l–1 ABA 1 week after veraison, and four of the blocks from each treatment were also sprayed at the same time with 500 mg l–1 CEPA at 2,000 l ha–1. The percentages of clusters having a medium or dark colour were assessed at harvest. Timing of ABA application Clusters were sampled four times during the season, beginning 1 week after veraison on 5 July. Breaker-stage berries with their pedicel were removed from the clusters and incubated with 0, 200, 400, or 600 mg l–1 ABA. Anthocyanin concentrations were determined after 7 d. The effect of the timing of ABA application in the vineyard was determined by spraying the clusters with 600 mg l–1 ABA at 1-week intervals. The experiment used four blocks per treatment and started on 2 July (veraison) until 6 August (= six weekly sprays). The percentages of medium and dark clusters were scored on 27 August. Effect of temperature on the response to ABA application The effect of high temperature before or after the application of ABA was examined in three experiments. Berry clusters were harvested 2 – 4 weeks after veraison and placed in growth chambers for 24 h at 18ºC, 25ºC, or 38ºC, at a relative humidity (RH) of 95%. The clusters were dipped in 0 or 600 mg l–1 ABA before or after incubation. Green and breaker-stage berries were then removed from the clusters, with their pedicels attached, and anthocyanin concentrations were determined after 7 d. Data are presented for one experiment which was representative of the response.

Statistical analysis Statistical analysis was performed using the JMP 7.0.1 platform (SAS Institute Inc., Cary, NC, USA) by one-way ANOVA with the LS Means Tukey-Kramer HSD protocol at P ≤ 0.05. For anthocyanin concentrations in the detached berry experiments, each treatment had three replicates, each with four to five berries. Colour evaluations of berry clusters in the vineyard measured 25 clusters in each of four replicates for each treatment. Where experiments were repeated more than once, the results were pooled and analysed together, except for the high temperature experiment.

RESULTS Effect of GA3 and ABA on colour development In both detached green and breaker-stage berries, ABA increased anthocyanin content compared with control berries (Figure 1A, B). In contrast, there was no response to GA3. The two berry stages showed similar responses. Similarly, in the vineyard, ABA increased the percentage of berries rated medium- or dark-coloured, with no effect of GA3 (Figure 2).

ABA (mg l–1) FIG. 2 Effect of ABA (at 200 or 400 mg l–1) and GA3 (at 0 or 15 mg l–1) on colour development in ‘Crimson Seedless’ grapes grown in the field. Grape clusters were untreated or treated with 15 mg l–1 GA3 at 6-mm berry diameter, then sprayed with ABA at veraison and rated for colour development at harvest. Different lower-case letters above each bar indicate that the means are significantly different according to the Tukey-Kramer test (P ≤ 0.05).

berries, CEPA had no effect on the accumulation of anthocyanins, while CEPA and ABA applied together increased anthocyanin concentrations similar to ABA alone (Figure 4). In contrast, CEPA increased colour development when it was applied in the vineyard (Figure 5). This response occurred with or without ABA.

Anthocyanin content (A530 g–1 FW)


Effect of ethylene on colour development In detached berry Experiment I, ABA typically increased anthocyanin concentrations in both green and breaker-stage berries, whereas ethylene and 1-MCP generally had no significant (P ≤ 0.05) effect (Figure 3A, B). In detached berry Experiment II, with breaker-stage

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Medium + dark berry clusters (%)

R. OVADIA, M. OREN-SHAMIR, T. KAPLUNOV, Y. ZUTAHY, A. LICHTER and S. LURIE


ABA (mg l–1)


ABA (mg l–1)

ABA (mg l–1) ABA (mg l–1)

FIG. 1 Effects of ABA and GA3 on the accumulation of anthocyanins in detached ‘Crimson Seedless’ grapes. GA3 was applied at 15 mg l–1 when the berries were 6-mm in diameter. ABA was then applied at 200 or 400 mg l–1 to detached green (Panel A) or breaker-stage (Panel B) berries. Data were recorded 7 d after the application of ABA. Different lowercase letters above each bar in each Panel indicate that the means are significantly different according to the Tukey-Kramer test (P ≤ 0.05).

FIG. 3 Effects of ABA, ethylene, and/or 1-MCP on the accumulation of anthocyanins in ‘Crimson Seedless’ grape berries. Grape clusters, harvested after the beginning of veraison, were untreated or treated with 100 µl l–1 ethylene, 2 µl l–1 1-MCP, or a combination of both, for 24 h. Detached berries were then treated with 0, 50, 200, or 400 mg l–1 ABA, and kept in a controlled climate chamber for 7 d, after which anthocyanin concentrations were determined. Data were from detached berries at the green-stage (Panel A) or the breaker-stage (Panel B). Different lower-case letters above each bar in each Panel indicate that the means are significantly different according to the Tukey-Kramer test (P ≤ 0.05).




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Date of ABA application FIG. 4 Effects of ABA and/or CEPA (2-chloroethylphosphonic acid) on the accumulation of anthocyanins in ‘Crimson Seedless’ grape berries. Clusters were dipped in 0, 0.2, 0.4, or 0.6% (v/v) CEPA and detached berries at the breaker-stage were then treated with 50 mg l–1 ABA. Anthocyanin concentrations were determined on day-0, and after 7 d in treated berries. Different lower-case letters above each bar indicate that the means are significantly different according to the Tukey-Kramer test (P ≤ 0.05).

Effect of high temperature on the response to ABA There were mixed responses to temperature and ABA, depending on when the berries were incubated, and whether the berries were at the green or breakerstage (Figure 8). In green berries, ABA was effective at 18ºC and 38ºC, when applied before the high temperature incubation. In contrast, ABA was ineffective when applied to berries after they had been incubated. In breaker-stage berries, ABA was effective at all temperatures when applied before incubation. However, there was a different response when ABA was applied after high temperature incubation, when ABA was effective only after the 18ºC treatment.

DISCUSSION Many seedless grape cultivars are sprayed with GA3 after flowering in order to increase berry size. The consensus is that there is no direct effect of GA3 on berry sugar content or colour (Davies and Böttcher, 2009), although delays in sugar accumulation (Guelfat-



Effect of timing of ABA application on colour development In detached berries, the optimum time to spray ABA depended on the concentration used (Figure 6). The best time to apply the low dose (200 mg l–1 ABA) was on 4 August (4 weeks after veraison began), the best times to apply the intermediate dose (400 mg l–1 ABA) were on 23 July and 4 August, while the highest dose (600 mg l–1) was equally effective at all times. The maximum response occurred followed the application of 200 mg l–1 ABA on 4 August. In the vineyard experiment which used a spray of 600 mg l–1 ABA, the best response to ABA occurred on 16 July (2 weeks after veraison), with poorer responses following earlier or later applications (Figure 7). The optimum treatment produced more than three-times the percentage of clusters rated medium- or dark- coloured than the untreated control.

FIG. 6 Effect of the timing of a dip application of ABA at 0, 200, 400, or 600 mg –1 l on the accumulation of anthocyanins in detached ‘Crimson Seedless’ grape berries. Veraison started on 5 July. Berry clusters were harvested on four different dates. Detached berries at the breaker-stage were treated with ABA and their anthocyanin concentrations were determined after 7 d. Different lower-case letters above each bar indicate that the means are significantly different according to the Tukey-Kramer test (P ≤ 0.05).

ABA (mg l–1) FIG. 5 Effects of ABA and CEPA (2-chloroethylphosphonic acid) on colour development in ‘Crimson Seedless’ grape berries. ABA (0, 200, or 400 mg l–1) and CEPA (500 mg l–1) were applied to the vines in a vineyard at veraison. CEPA was only applied to the control (0) and 200 mg l–1 ABA treatments. At harvest, the grape clusters were rated for the percentage of clusters with medium- and dark-coloured berries. Different lowercase letters above each bar in each panel indicate that the means are significantlydifferent according to the Tukey-Kramer test (P ≤ 0.05).

Date of ABA application FIG. 7 Effect of the timing of an application of ABA in the vineyard on colour development in ‘Crimson Seedless’ grape berries. Clusters were treated with 600 mg l–1 ABA in the vineyard on six different dates, beginning at varaison on 2 July. Colour ratings of the clusters were made at harvest on 27 August and expressed as the percentage of clusters having medium- and dark-coloured berries. Different lower-case letters above each bar in each panel indicate that the means are significantly different according to the Tukey-Kramer test (P ≤ 0.05).

0 mg l–1 ABA 600 mg l–1 ABA



R. OVADIA, M. OREN-SHAMIR, T. KAPLUNOV, Y. ZUTAHY, A. LICHTER and S. LURIE

FIG. 8 Effect of a high-temperature treatment before or after the application of ABA, on the accumulation of anthocyanins in ‘Crimson Seedless’ grape berries treated with 600 mg l–1 ABA before or after each temperature treatment. ABA → Temperature, indicates that the ABA was applied before the temperature treatment. Temperature → ABA, indicates that the clusters were first held at the temperature indicated, then dipped in ABA. The temperature treatments (18ºC, 25ºC, and 38ºC) were applied in controlled temperature and humidity chambers for 24 h. Detached berries from clusters at the green-stage (Panel A) or the breaker-stage (Panel B) were then held for 7 d at 20ºC, then the anthocyanin concentrations of the berry skins were measured. Lower-case letters above each bar indicate that the means for each treatment are significantly different according to the Tukey-Kramer test (P ≤ 0.05).

Reich and Safran, 1973) and in anthocyanin content have been reported (Han et al., 1996). The latter was probably due to an indirect effect of GA3 on the growth rate of the berries (Davies and Böttcher, 2009). In the present study, GA3 had no effect on the promotion of colour development by ABA in grape berries. Three plant growth substances are associated with grape berry maturation: ABA, ethylene, and brassinosteroids (Davies and Böttcher, 2009). There are many examples of positive responses in berry colour to CEPA, which releases ethylene (e.g., El-Kereamy et al., 2003), but there are also many examples of no effect. The application of ethylene gas was suggested to act via a different route from ABA, according to a lack of response of MybA which is a major regulator of normal colour development (Tira-Umphon et al., 2007). In the current study, CEPA enhanced berry colour development in the vineyard, whereas CEPA or ethylene gas had no effect after harvest. One possible explanation is that ethylene affects the flux of assimilates associated with berry ripening, including sugar accumulation (Chervin et al., 2004; 2006; Mailhac and Chervin, 2006). The leaves adjacent to the clusters are the primary target for field spray applications. Therefore, detached berries can be considered an

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abnormal target for ethylene sprays. In contrast, postharvest application of ethylene to ‘Aleatico’ grapes prevented the loss of anthocyanins after 13 d at 20ºC, while untreated grapes lost 25% of their anthocyanins (Bellincontro et al., 2006). The present study kept the detached berries for 7 d, and did not show any decrease in anthocyanin concentrations. Endogenous ABA accumulates at veraison and affects berry maturation (Davies and Böttcher, 2009; Conde et al., 2007). These findings are supported by the results from vineyard studies which showed a consistent effect of ABA on colour development (Cantin et al., 2007; Peppi et al., 2007a; Peppi and Fidelibus, 2008). Likewise, consistent effects of ABA were observed in a previous study with detached grapes (Lurie et al., 2009), and in this study. In most studies, ABA was applied at veraison. In an experiment with ‘Flame Seedless’ grapevines, applications were made pre-veraison, at veraison, and post-veraison (Peppi et al., 2006). The best time for ABA application was found to be at veraison, although a spray 2 weeks after veraison also increased berry colour. In the present study, the best time to apply ABA was 2 weeks after veraison, but the benefits were also observed for a further 4 weeks. In detached berries, the response to ABA changed over time. The maximum response to ABA was found 3 – 4 weeks after veraison. Interestingly, high concentrations of ABA applied to detached berries were either not effective, or inhibited colour development compared to a low concentration. Responsiveness to ABA was maintained in mature berries, as has been reported by Peppi et al. (2007b). Day and night temperatures > 30ºC prevented the accumulation of anthocyanins in high or low light; while, at 24ºC, more anthocyanins were produced in high light than in low light (Kliewer, 1977). ‘Pinot Noir’ grape berries grown at 30ºC or 35ºC had lower levels of the mRNA for flavonoid-3’,5’-hydrolase in their skin, suggesting that changes in anthocyanin accumulation were regulated by transcription of this gene (Mori et al., 2007). In the field, exposure of grapes to high temperatures is generally transient, with generally low temperatures at night. However, heat-waves can increase night temperatures. In the experiment with detached berries, we found that the response was similar at 18ºC, 25ºC, or 38ºC when berries were incubated after being treated with ABA. Temperatures below 20ºC enhanced the production of ABA and, therefore, anthocyanin accumulation (Yamane et al., 2006). However, it has also been reported that a temperature of 36ºC or 38ºC for 4 h increased the concentration of both free and bound ABA in grapevine leaves and in cultured cells (Abass and Rajashekar, 1993). It is likely that transient heat stress does not limit anthocyanin production.

CONCLUSIONS Exogenous ABA promoted colour development in ‘Crimson Seedless’ grape berries, both in the vineyard and in detached clusters. Early applications of GA3 to encourage berry growth, or a high temperature for 1 d, did not affect the response to ABA. The response to

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ABA was optimal when the chemical was applied 2 – 3 weeks after the start of veraison. Although grapes responded to CEPA in the vineyard, there was no response in detached berries. These results indicate that

‘Crimson Seedless’ grapevines grown in a warm climate, where colour development is poor, would benefit from a spray of 200 – 400 mg l–1 ABA, 2 – 3 weeks after the start of veraison.

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